GABA is an important neurotransmitter in vertebrates and insects; GABA receptors form major targets for drugs in man and for pesticides in invertebrates. Despite the recent molecular cloning of vertebrate GABA receptors, the insect GABA receptor remains uncloned and poorly understood. Cloning of this receptor in Drosophila melanogaster will allow its study via the advanced manipulative genetics available in this model organism. Further, increased understanding of this receptor will allow for the rationale design of novel insecticides active at this site. Insecticides with new modes of action are urgently needed to counter the growing number of multiply resistant disease vectors such as malarial mosquitoes. Cyclodienes are GABA antagonists acting at binding sites on the GABA receptor. Our isolation of a cyclodiene insensitive mutant of Drosophila melanogaster thus represents a unique opportunity to clone a putative Insect GABA receptor. We have previously localized this gene to a single lettered sub-region on the polytene chromosome. The objectives of this project are thus to clone and analyze this gene and thereby obtain an enhanced understanding of GABA receptor structure and function. The gene will be cloned through the precise knowledge of its location via a chromosomal walk. The gene will be located within the walk by the combined approaches of deficiency breakpoint analysis, P-element mutagenesis and oligonucleotide probing. Confirmation of gene cloning will be achieved by the rescue of sensitivity following transformation of insensitive flies, by P-element mediated germline transformation, with a copy of the sensitive gene. Following cloning, the gene will be sequenced and its amino acid sequence predicted. The relationship of gene structure to function will be elucidated via a number of approaches. 1) Sequence comparison of a number of different alleles. 2) Functional expression of sensitive and insensitive cDNA's in Xenopus oocytes and a comparison of their pharmacology. 3) Functional expression of cDNA's following in vitro mutagenesis and an examination of the events leading to ligand insensitivity or loss of function.
